T cell factor 1 (Tcf1) is essential for T cell development; however, it remains controversial whether β-catenin, a known coactivator of Tcf1, has a role. Tcf1 is expressed in multiple isoforms in T lineage cells, with the long isoforms interacting with β-catenin through an N-terminal domain. In this study, we specifically ablated Tcf1 long isoforms in mice (p45−/−mice) to abrogate β-catenin interaction. Although thymic cellularity was diminished in p45−/− mice, transition of thymocytes through the maturation stages was unaffected, with no overt signs of developmental blocks. p45−/− thymocytes showed increased apoptosis and alterations in transcriptome, but these changes were substantially more modest than in thymocytes lacking all Tcf1 isoforms. These data indicate that Tcf1–β-catenin interaction is necessary for promoting thymocyte survival to maintain thymic output. Rather than being dominant-negative regulators, Tcf1 short isoforms are adequate in supporting developing thymocytes to traverse through maturation steps and in regulating the expression of most Tcf1 target genes.
T lymphocytes constitute the cellular branch of the adaptive immune response and are essential for clearing infections by viruses, bacteria, and parasites. T cells are generated in the thymus, following step-wise maturation stages. The earliest step is the seeding of hematopoietic stem cell–derived progenitors in the thymus, including the early thymic progenitors that undergo T cell lineage specification and commitment steps within the CD4−CD8− double-negative (DN) stages. Based on CD44 and CD25 expression, DN thymocytes can be divided into four sequentially developing subsets: CD44+CD25− DN1, CD44+CD25+ DN2, CD44−CD25+ DN3, and CD44−CD25− DN4 cells. DN4 cells then pass through CD8+ immature single-positive stage and become CD4+CD8+ double-positive (DP) thymocytes. After vigorous negative and positive selection, the DP thymocytes make lineage choice decisions to become CD4+ or CD8+ single-positive (SP) cells. Each of these maturation steps is orchestrated by multiple transcription factors in different combinations (1, 2).
T cell factor 1 (Tcf1) has been known as a transcription factor acting downstream of the Wnt pathway. It can interact with β-catenin coactivator, which is posttranslationally regulated and stabilized by Wnt- and PG-derived signals (3, 4). Tcf1 critically regulates several stages of thymic T cell development. At the very early step, Tcf1 is potently upregulated by Notch signaling and contributes to commitment of early progenitors to the T cell lineage (5, 6). Although Tcf1 is not absolutely essential for TCRβ locus rearrangements at the DN3 stage, it facilitates maturation of DN to DP thymocytes (7). In recent studies, we demonstrated that Tcf1 is important for directing DP thymocytes to the CD4+ T cell lineage by acting upstream of Thpok (8). Although not required for the CD8+ T cell lineage decision, Tcf1 uses its intrinsic histone deacetylase (HDAC) activity to suppress the expression of CD4+ lineage-associated genes and, hence, establish CD8+ T cell identity (9).
As the result of differential promoter usage and alternative splicing, multiple Tcf1 isoforms can be detected in T cells (10). All isoforms contain a C-terminal high mobility group (HMG) DNA-binding domain and a newly discovered HDAC domain (9). The Tcf1 long isoforms (p45 and p42) contain a unique N-terminal β-catenin–binding domain, whereas the Tcf1 short isoforms (p33 and p30) lack this domain and, hence, cannot interact with β-catenin. Most of the loss-of-function studies of Tcf1 ablated all Tcf1 isoforms. The Tcf1 short isoforms have been considered dominant-negative regulators of the full-length Tcf1 protein, yet their physiological roles in T cell development have not been elucidated. In this study, we performed structure-function analysis of Tcf1 protein in developing thymocytes by ablating the β-catenin–binding domain or the HMG DNA-binding domain (Fig. 1A).
Materials and Methods
Mice and CRISPR/Cas9-mediated genome editing
Tcf7fl/fl and p45−/− mice were generated previously (10, 11). To generate Tcf1ΔHMG mutant mice, we used the CRISPR/Cas9 approach (12) to insert a stop codon in Tcf7 exon 8 after the codon encoding Val304. Briefly, a single guide RNA ([sgRNA]: 5′-GAGGGGTTTCTTGATGAC-3′) was cloned into an sgRNA vector using OriGene’s gRNA Cloning Service (Rockville, MD) and was used as a template to synthesize sgRNAs using the MEGAshortscript T7 Kit (Life Technologies). Cas9 mRNA was transcribed in vitro using the mMESSAGE mMACHINE T7 Ultra Kit (Life Technologies), with plasmid MLM3613 (catalog no. 42251; Addgene) as a template. Next, Cas9 mRNA (100 ng/μl) was mixed with sgRNA (50 ng/μl) and 100 ng/μl oligonucleotides (5′-TCT CAT CTC CTT CAT GTA AAG CAT GAA CGC ATT GAG GGG TTT CTT GAT CTA GAC TGG CTT CTT AGC CTC CTT CTC TGC CTT GGG TTC TGC CTG TGT TTT-3′) and microinjected into the cytoplasm of fertilized eggs collected from C57BL/6N inbred mice. The injected zygotes were surgically implanted into the oviducts of pseudo-pregnant foster mothers, and offspring were genotyped using PCR and DNA sequencing. All mice analyzed were 5–9 wk of age, and both genders were used without randomization or blinding. All mouse experiments were performed under protocols approved by the Institutional Animal Use and Care Committees of the University of Iowa and the National Heart, Lung, and Blood Institute.
Flow cytometry and apoptosis assay
Single-cell suspensions were prepared from the thymus and surface stained as described (7). The following fluorochrome-conjugated Abs were used: anti-TCRβ (H57-597), anti-CD8 (53–6.7), anti-CD4 (RM4-5), anti-CD44 (IM7), anti-CD62L (MEL-14), and anti-CD25 (PC61.5) (all from eBioscience). Cell apoptosis was detected with a PE Annexin V Apoptosis Detection Kit (BD Biosciences). Hoechst 33258 was used to exclude dead cells so as to specifically measure Annexin V+ apoptotic cells. Data were collected on a FACSVerse (BD Biosciences) and analyzed with FlowJo software (TreeStar).
Cell lysates were prepared from the total thymocytes and probed with anti-Tcf1 (C46C7), anti-Lef1 (C18A7) (both from Cell Signaling Technology), or anti–β-actin (I-19; Santa Cruz Biotechnology). The Tcf1 Ab is produced by immunizing animals with a synthetic peptide corresponding to a region surrounding Leu158 of human Tcf1 protein; it recognizes all Tcf1 isoforms and would detect Tcf1ΔHMG truncated protein if it were translated and stably expressed.
RNA sequencing and data analysis
DN3 thymocytes were sorted from control, p45−/−, or Vav-Cre+Tcf7fl/fl (Tcf1−/−) mice, and total RNA was extracted as described (7). cDNA libraries were prepared and sequenced on an Illumina HiSeq 2000 in single-read mode, with a read length of 50 nt. The single-end fastq reads were assessed for quality using FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/); adaptor and low-quality regions were trimmed using Trimmomatic. Reads were then aligned to the mm9 mouse reference sequence using TopHat2, and read counts were obtained using featureCounts (13). Differential expression was determined with DESeq2 using a custom R script (14). Heat maps were created using standard R tools by plotting the log2 regularized log transformation. RNA sequencing (RNA-Seq) data were submitted to the Gene Expression Omnibus under accession number GSE92323 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?&acc=gse92323).
Results and Discussion
Tcf1 short isoforms adequately support thymic maturation transitions
Tcf1 proteins are encoded by the Tcf7 gene, with long isoforms produced from transcripts initiated at exon 1 and short isoforms produced from transcripts initiated at exon 3. Previously, we generated a Tcf1-EGFP reporter mouse strain in which we inserted an IRES-EGFP cassette into the first intron of the Tcf7 gene (11). In front of the cassette, we placed an En2 splice acceptor that forced the splicing of exon 1 to this cassette instead of exon 2. Thus, mice homozygous for the Tcf1-EGFP reporter alleles failed to produce the Tcf1 long isoforms (p45 and p42) but retained the short isoforms (p33 and p30), albeit at lower levels than those in wild-type (WT) mice or heterozygotes (Fig. 1B); for simplicity, this strain is called p45−/− herein. Importantly, Lef1 proteins (in two isoforms) were not noticeably affected in p45−/− thymocytes (Fig. 1B).
Loss of the Tcf1 long isoforms reduced the total thymic cellularity by ∼60% (Fig. 1C). By phenotypic analysis, p45−/− mice showed profiles of thymic maturation stages that were similar to WT or p45+/− mice, with a modest decrease in the DP frequency and a concurrent mild increase in the frequency of CD4+ and CD8+ SP thymocytes (Fig. 1D, Supplemental Fig. 1A). As a result of the reduction in total thymic cellularity, all thymic subsets in p45−/− mice were reduced in numbers (Supplemental Fig. 1A). As for early stages, p45−/− DN thymocytes showed a modest, but consistent, decrease in the DN2 subset, in both frequency and numbers, whereas other DN subsets were similar among WT, p45+/−, and p45−/− mice (Fig. 1D, Supplemental Fig. 1B). For non-T lineage thymocytes, thymic B cells, dendritic cells, NK cells, and group 1 innate lymphoid cells were detected at increased frequencies in p45−/− mice, but their absolute counts were not altered significantly (Supplemental Fig. 1C–E). It is known that complete deletion of all Tcf1 isoforms diminishes total thymic cellularity to <5 million cells and results in several incomplete developmental blocks at the DN1 and/or DN3 stages (15) (also see below). Our analysis of p45−/− mice suggests that Tcf1 short isoforms, even at reduced levels, are adequate to support the normal transition among thymocyte maturation stages. Nevertheless, Tcf1 long isoforms remain important for optimal thymic cellularity.
Tcf1 long isoforms are necessary for thymocyte survival
Germline targeting of Tcf1 has detrimental effects on T cell development (15, 16). We previously established a Tcf7fl/fl strain (10), and crossed it with Vav-Cre mice to specifically ablate all Tcf1 isoforms in hematopoietic cells. Vav-Cre+Tcf7fl/fl mice (Tcf1−/−) showed a phenocopy of the germline-targeted strain, exhibiting greatly reduced thymic cellularity (Fig. 2A), a strong block at the DN stage, a corresponding reduction in DP thymocytes, and a skewed CD4+ to CD8+ T cell lineage distribution (Fig. 2B, Supplemental Fig. 2A). Within the DN population, Tcf1−/− mice showed loss of DN2 thymocytes and accumulation of DN1 and DN3 thymocytes, and Tcf1−/− DN1 cells exhibited an aberrant upregulation of CD25 (Fig. 2B, Supplemental Fig. 2A), consistent with previous reports (15).
A prominent role for Tcf1 is to ensure survival of early thymocytes at the DN and DP stages (17, 18). In line with previous reports, ablation of all Tcf1 isoforms/proteins caused strong apoptosis of DP, CD4+, and mature CD8+ SP thymocytes (Supplemental Fig. 2B). In addition, all Tcf1−/− DN subsets were more apoptotic than controls (Fig. 2C, 2D). In contrast, p45−/− thymocytes exhibited increased apoptosis (with the exception of DP thymocytes) but at much more modest levels compared with Tcf1−/− cells, whereas p45+/− and Tcf1+/− thymocyte survival was not noticeably different from WT cells (Fig. 2C, 2D, Supplemental Fig. 2B). Collectively, these data indicate that Tcf1 long isoforms remain important for supporting thymocyte survival, which accounts, at least in part, for the reduced numbers of thymocytes at each developmental stage.
Loss of Tcf1 long isoforms minimally affects the DN3 transcriptome
We next investigated how a deficiency in Tcf1 long isoforms or all Tcf1 isoforms affected gene expression. Because Tcf1−/− mice showed a strong block at the DN3 stage, and p45−/− and Tcf1−/− DN3 cells showed increased apoptosis, we focused on DN3 thymocytes for RNA-Seq analysis, which showed good reproducibility between two replicates (Supplemental Fig. 2C). Using criteria of ≥1.5-fold expression changes and an adjusted p value < 0.05, we identified all differentially expressed genes (DEGs) in p45−/− versus control DN3 thymocytes and DEGs in Tcf1−/− versus control DN3 thymocytes. Unsupervised clustering analysis revealed that Tcf1−/− cells caused strong transcriptomic changes, whereas only a small portion of such changes was observed in p45−/− DN3 thymocytes (Fig. 3A). Among the 1044 upregulated genes, only 163 (15.6%) were upregulated in p45−/− DN3 cells (Fig. 3B). Functional annotation showed that Fasl (encoding Fas ligand) was among the 85 uniquely upregulated genes in p45−/− DN3 cells, and Gzma (encoding granzyme A) was induced in both p45−/− and Tcf1−/− DN3 cells (Fig. 3B). Another interesting gene is Dtx1 (encoding Deltex 1 downstream of the Notch signaling pathway), which was upregulated in p45−/− and Tcf1−/− DN3 cells, consistent with a role for Tcf1 in terminating Notch signals in committed T lineage cells (19) (Fig. 3B). Among the 1219 downregulated genes, only 91 (7.5%) were downregulated in p45−/− DN3 cells (Fig. 3C). Interestingly, Rag1 was among the 39 uniquely downregulated genes in p45−/− DN3 cells (Fig. 3C), although the physiological impact remains to be determined. Overall, deficiency in Tcf1 long isoforms had limited impact on the DN3 transcriptome compared with loss of all Tcf1 isoforms/proteins. This observation held true when DEGs were identified using more stringent criteria (Supplemental Fig. 2D). These data further suggest that the Tcf1 short isoforms are adequate to support most Tcf1 target gene expression in developing thymocytes.
C terminus truncation destabilizes Tcf1 transcripts and proteins
Recently, we demonstrated that Tcf1 has intrinsic HDAC activity that is mapped between the β-catenin–binding domain and the HMG-binding domain (9). We aimed to determine whether Tcf1 HDAC activity can be dissociated from its DNA-binding capacity. To this end, we used the CRISPR/Cas9 approach to insert a stop codon after Val304 in Tcf7 exon 8, thus leading to truncation of the HMG domain at the C terminus (Fig. 1A, called the Tcf1ΔHMG allele in this article). Although the heterozygotes had similar thymic cellularity as littermate controls, Tcf1ΔHMG/ΔHMG mice showed greatly reduced total thymocyte numbers (Fig. 4A). T cell development in Tcf1ΔHMG/ΔHMG mice showed a block at the DN stage, with a concomitant reduction in DP cells and a skewed CD4+/CD8+ ratio (Fig. 4B, Supplemental Fig. 2E). Furthermore, the DN cells in Tcf1ΔHMG/ΔHMG mice exhibited an accumulation of DN1 and DN3 thymocytes, as well as a loss of DN2 cells (Fig. 4B, Supplemental Fig. 2E). These profiles were remarkably similar to those in Tcf1−/− mice (compared with Fig. 2). By immunoblotting with a Tcf1 Ab that is generated with a peptide surrounding human Tcf1 Leu158 as an immunogen, we found that Tcf1ΔHMG/ΔHMG thymocytes were devoid of any Tcf1 protein (Fig. 4C). Unexpectedly, at the mRNA level, Tcf1 transcripts were greatly reduced in Tcf1ΔHMG/ΔHMG thymocytes and were even lower than those in Tcf1−/− cells (Supplemental Fig. 2F). Therefore, the absence of all Tcf1 proteins in Tcf1ΔHMG/ΔHMG mice might be ascribed to at least two possibilities: insertion of a stop codon in exon 8 destabilized the mRNA and/or any protein translated from the diminished amounts of mutant transcripts was not stable. This model does not achieve the intended dissociation of Tcf1 HDAC activity from DNA binding; nonetheless, it suggests that unnatural mutation(s) in the HMG domain strongly impairs the stability of Tcf1 mRNA and/or protein.
One unique feature of Tcf1 long isoforms is their ability to interact with the coactivator β-catenin. Forced expression of β-catenin extends thymocyte survival (20). However, conditional targeting of β-catenin neither enhanced thymocyte death (21) nor strongly diminished thymic cellularity (22). One possible explanation is that the existing β-catenin–targeted mouse strains retained a truncated form of β-catenin protein in hematopoietic cells (23), and the truncated protein might be partially functional. γ-Catenin is a homolog of β-catenin and interacts with Tcf4 at a region right next to the β-catenin–binding domain (24). γ-Catenin is expressed in T cells, and its interaction with Tcf1 should be limited to the long isoforms (22). Therefore, our new targeting approach in p45−/− mice ensured complete abrogation of Tcf1 interaction with β-catenin and/or γ-catenin. Thus, our data suggest that one major function of Tcf1–β-catenin interaction is to control thymocyte life span and sustain the output of mature T cells from the thymus. It should be noted that, in addition to a complete loss of Tcf1 long isoforms, p45−/− thymocytes expressed reduced amounts of Tcf1 short isoform proteins. The reduced amount of Tcf1 short isoforms could have contributed to the modest increase in the apoptosis of p45−/− thymocytes and further suggest that Tcf1–β-catenin interaction may also have a role in the proposed positive feed-forward regulation of Tcf1 gene transcription (6). Nonetheless, the latter possibility remains an integral part of Tcf1–β-catenin complex–mediated regulation of thymocyte survival.
Tcf1 short isoforms have been considered dominant-negative regulators or nonfunctional (6, 25). It was shown that a Tcf1 p45 transgene can partially restore thymocyte numbers and rectify T cell developmental defects in Tcf1-deficient mice; however, a Tcf1 p33 transgene failed to do so (17). In this study, instead of complementing Tcf1 deficiency using a transgene, we perturbed generation of Tcf1 long isoforms in the Tcf7 gene locus. Because Tcf1 can potentially regulate its own expression via a positive feed-forward loop (6), the expression of Tcf1 short isoforms appeared to be lower in p45−/− thymocytes (Fig. 1B). Despite the diminished expression, the Tcf1 isoforms were adequate to support the developing thymocytes to traverse through each maturation step without causing detectable blocks. In addition, Tcf1 short isoforms adequately support normal expression of the majority (>85%) of Tcf1-dependent genes in DN3 thymocytes. Thus, our findings suggest that the Tcf1 short isoforms are essential regulators of T cell maturation in the thymus rather than being dominant negative. Because Tcf1 has critical roles in regulating mature CD4+ and CD8+ T cell responses (4), it would be of interest to further investigate whether Tcf1 short isoforms are adequate in directing the generation of central memory CD8+ T cells and/or the differentiation of follicular helper CD4+ T cells.
We thank the University of Iowa Flow Cytometry Core facility (J. Fishbaugh and H. Vignes) for cell sorting and Igor Antoshechkin (California Institute of Technology) for RNA-Seq.
This work was supported by grants from the National Institutes of Health (AI112579, AI115149, AI119160, and AI121080 to H.-H.X.) and the U.S. Department of Veterans Affairs (I01 BX002903 to H.-H.X.). C.L. is supported by the intramural research program of the National Heart, Lung, and Blood Institute, National Institutes of Health.
RNA sequencing data presented in this article have been submitted to the Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo) under accession number GSE92323.
The online version of this article contains supplemental material.
Abbreviations used in this article:
differentially expressed gene
high mobility group
single guide RNA
T cell factor 1
The authors have no financial conflicts of interest.